Physical Studies of Conformational Plasticity in a Recombinant Prion Protein

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Abstract
PrPSc is known to be the major, if not the only, component of the infectious prion. Limited proteolysis of PrPSc produces an N-terminally truncated polypeptide of about 142 residues, designated PrP 27−30. Recently, a recombinant protein (rPrP) of 142 residues corresponding to the Syrian hamster PrP 27−30 was expressed in Escherichia coli and purified (Mehlhorn et al., 1996). rPrP has been refolded into both α-helical and β-sheet structures as well as various intermediates in aqueous buffers. The β-sheet state and two pH-dependent α-helical states were characterized by CD and NMR. The α-helical conformation occurred only after the formation of an intramolecular disulfide bond, whereas the β-sheet form was accessible either with or without the disulfide. Of the different α-helical forms studied, only those refolded in the pH range 5−8 were substantially soluble at physiological pH, exhibiting similar conformations and monomeric analytical sedimentation profiles throughout the above pH range. Furthermore, refolded α-rPrP showed NMR chemical shift dispersion typical of proteins with native conformations, although 2D NMR indicated large segments of conformational flexibility. It displayed a cooperative thermal denaturation transition; at elevated temperatures, it converted rapidly and irreversibly to the thermodynamically more stable β-sheet form. Unfolding of α-rPrP by GdnHCl revealed a two-phase transition with a relatively stable folding intermediate at 2 M GdnHCl. The ΔG values were estimated to be 1.9 ± 0.4 kcal/mol for the first phase and 6.5 ± 1.2 kcal/mol for the second, consistent with a folding core surrounded by significant segments of flexible conformation. By NMR, α-rPrP(acid) isolated at pH 2 without refolding exhibited heterogeneous line widths, consistent with an acid-denatured molten globular state. We conclude that to the extent that rPrP constitutes a relevant folding domain of PrPC, the various conformations exhibited by rPrP suggest that the PrP sequence may be intrinsically plastic in its conformations; indeed, portions of PrPC may possess a relatively open conformation which makes it susceptible to conversion into PrPSc under appropriate conditions.